Free radical damage to DNA, which has been associated with carcinogenesis, the normal aging process, and neurodegeneration, produces a variety of lesions, including various oxidized abasic sites (OAS). These include 1'-oxidized, 4'-oxidized or fragmented deoxyribose residues, known products of agents such as H2O2, ionizing radiation, and bleomycin. Such damage have cytotoxic and mutagenic potential that might underlie the age and disease effects cited above, but OAS have been difficult to measure specifically, sensitively and quantitatively by existing methods. This project will capitalize on its initial success to continue development of and to apply new methods for such analysis, with an emphasis on the use of DNA repair enzymes to liberate specific damages for detection by advanced techniques of mass spectrometry. These methods will be used to explore the formation and repair of individual OAS in vivo. Abasic (AP) endonucleases are implicated in the repair of these damages, but this specifically has not been demonstrated in vivo. We will examine whether there are defects in repair of OAS in AP endonuclease-deficient bacteria, yeast and, if available, mammalian cells. The repairability in vitro and mutagenic potential in vivo of a specific OAS will be assessed using a synthetic, photosensitive 1'-t-butyl ketone derivative to target the damage in specific sequences. This work will be important for future work toward understanding the role of free-radical damage in aging, cancer, and neurodegeneration, and it will substantially improve the methodology for molecular analysis of this important class of oxidative DNA damage.